Methods and means for diagnosing lung cancer

Abstract

The present invention relates to the diagnosis of lung tumors. It provides methods suitable both for diagnosing lung tumors on the basis of surgical samples and lung biopsies (here, e.g., with the aid of DNA microarrays) and of liquid biopsies. In the case of liquid biopsies, cell-free DNA (cfDNA) is used. In this context, both particularly suitable analysis methods and particularly suitable sets of methylation markers are described. Means suitable for diagnosing lung cancer by examinining the methylation of a set of methylation markers, e.g., in cell-free DNA (cfDNA) from liquid biopsy samples of patients, wherein the means comprises oligonucleotides which can hybridize to DNA comprising the methylation markers, as well as the use of said methods and means for diagnosing, i.e., e.g., determinination, subtyping and prognostic characterization of lung tumors are also an object of the invention.

1 . A method comprising determining the methylation of a set of methylation markers in a sample from a patient, wherein the set of methylation markers is selected from the group consisting of the regions listed in Table 1a, 1b and 1c and comprises at least 60 regions. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the set of methylation markers comprises at least 60 regions selected from the group consisting of: chr1(6165201-6165361), chr1 (17567892-17568189), chr1 (15426262-15426418), chr115670403-15670539), chr2 (1126410-1126557), chr2 (225642009-225642217), chr2 (236745514-236745688), chr2 (240881986-240882138), chr2 (2179742-2179886), chr2 (30747398-30747539), chr2 (175998270-175998415), chr2 (219647407-219647560), chr3 (56445240-56445378), chr3 (85143433-85143600), chr3 (146123966-146124095), chr3 (68947379-68947542), chr3 (197767819-197767978), chr4 (143487129-143487273), chr4 (26398190-26398329), chr4 (77647893-77648027), chr4 (102497551-102497732), chr5 (39187156-39187287), chr5 (56145736-56145896), chr5 (160171748-160171896), chr5 (16793080-16793219), chr5 (76869108-76869253), chr6 (169050287-169050447), chr6 (76773251-76773422), chr6 (123869831-123869971), chr7 (6268960-6269087), chr7 (38508407-38508486), chr7 (153743779-153743947), chr7 (137230794-137230963), chr7 (151300131-151300282), chr8 (3672236-3672387), chr8 (99510084-99510252), chr8 (101170822-101170975), chr8 (141127042-141127183), chr9 (2050654-2050804), chr9 (9227683-9227824), chr9 (79060522-79060633), chr9 (124334690-124334848), chr9 (126166694-126166828), chr10 (96279972-96280055), chr10 (97033594-97033733), chr11 (134245966-134246129), chr12 (8004422-8004573), chr12 (97140774-97140905), chr12 (111566555-111566698), chr12 (117750775-117750937), chr13 (36828740-36828902), chr14 (93214072-93214242), chr15 (56006471-56006552), chr15 (101547384-101547527), chr16 (4141795-4141956), chr18 (21857621-21857750), chr18 (29528340-29528468), chr18 (46845901-46846043), chr19 (874766-874934), chr19 (6799968-6800095), chr20 (20243607-20243747), chr20 (55079800-55079945), chr21 (30502729-30502871), and chr21 (46587906-46588052) 
 wherein the presence of a tumor is analyzed, wherein the set of methylation markers optionally comprises all regions of the group.   
     
     
         6 . The method of  claim 5 , wherein the set of methylation markers comprises at least 340 regions selected from the group consisting of the regions listed in Table 1a. 
     
     
         7 . The method of  claim 1 , wherein the set of methylation markers comprises at least 134 regions selected from the group consisting of: chr1 (3289010-3289139, chr1 (17567892-17568189), chr1 (23284417-23284507), chr1 (24277975-24278154), chr1 (47738990-47739142), chr1 (79467955-79468081), chr1 (108975333-108975476), chr1 (196682870-196683025), chr1 (217310510-217310654), chr1 (240656480-240656649), chr1 (240746545-240746706), chr1 (246241918-246242056), chr2 (1129413-1129596), chr2 (1334513-1334640), chr2 (23917010-23917136), chr2 (25124037-25124165), chr2 (46779214-46779381), chr2 (113534514-113534653), chr2 (120417931-120418073), chr2 (131798797-131798977), chr2 (198073787-198073950), chr2 (205889570-205889704), chr2 (207319476-207319691), chr3 (3755582-3755730), chr3 (14959981-14960128), chr3 (25581721-25581859), chr3 (75834579-75834736), chr3 (87031909-87032079), chr3 (122710736-122710872), chr3 (139727561-139727706), chr3 (145864433-145864574), chr4 (1665996-1666155), chr4 (22518120-22518271), chr4 (77306769-77306948), chr4 (82520036-82520212), chr4 (155413871-155414011), chr4 (156601279-156601436), chr4 (162457724-162457860), chr4 (176636441-176636580), chr4 (177654193-177654363), chr5 (14450118-14450272), chr5 (75935318-75935450), chr5 (140475728-140475872), chr5 (146345906-146346062), chr5 (156458027-156458167), chr5 (157169890-157170038), chr6 (20832000-20832349), chr6 (24420281-24420413), chr6 (36331071-36331215), chr6 (54074847-54075021), chr6 (71122323-71122483), chr6 (83604672-83604779), chr6 (90709859-90710016), chr6 (111744738-111744881), chr6 (148806765-148806922), chr6 (155574119-155574263), chr6 (158460178-158460323), chr7 (5549605-5549675), chr7 (40669616-40669796), chr7 (73799798-73799908), chr7 (78030021-78030155), chr7 (81399230-81399365), chr7 (134452355-134452524), chr7 (140335200-140335344), chr7 (146925646-146925824), chr7 (153976496-153976643), chr7 (157941162-157941344), chr7 (157980130-157980264), chr7 (157980485-157980624), chr7 (158314155-158314301), chr8 (6392188-6392336), chr8 (11724061-11724159), chr8 (17237496-17237639), chr8 (21803649-21803801), chr8 (52696850-52697008), chr8 (72183950-72184120), , hr8 (81042553-81042694), chr8 (85101824-85101952), chr8 (110703169-110703320), chr8 (121727803-121727944), chr8 (133476418-133476558), chr9 (8813022-8813150), chr9 (90258110-90258253, chr9 (97061691-97061835), chr10 (12533631-12533768), chr10 (32657588-32657719), chr10 (37511104-37511239), chr10 (62708104-62708269), chr10 (73207931-73208064), chr10 (108812804-108812940), chr10 (115658133-115658275), chr10 (123914649-123914808), chr11 (15025357-15025499), chr11 (19778770-19778909), chr11 (26355535-26355711), chr11 (26600784-26600925), chr11 (26626367-26626558), chr11 (41275397-41275536), chr11 (62158845-62158985), chr11 (70503001-70503139), chr11 (106592142-106592304), chr11 (120644150-120644282), chr11 (122678508-122678636), chr11 (128851150-128851286), chr12 (125571801-125571933), chr13 (48806444-48806588), chr13 (113527733-113527876), chr14 (104486171-104486314), chr15 (22839905-22840043), chr15 (26964926-26965065), chr15 (29246303-29246447), chr15 (30180680-30180842), chr15 (32404970-32405130), chr15 (64244033-64244215), chr15 (68530927-68531091), chr15 (83579367-83579513), chr15 (88559865-88560003), chr16 (6257325-6257474), chr16 (15665564-15665721), chr16 (24321180-24321320), chr16 (75528556-75528698), chr16 (88013993-88014135), chr16 (89713952-89714124), chr17 (416719-416865), chr17 (19809670-19809830), chr17 (21086965-21087112), chr17 (33364961-33365040), chr17 (64330485-64330837), chr17 (75142732-75142885), chr19 (11890923-11891074), chr19 (49016450-49016584), chr19 (57922060-57922195), chr20 (9706282-9706429), chr20 (33713618-33713757), chr21 (33340955-33341038), chr22 (21206849-21206995), chr22 (30292326-30292475), and chr22 (35697444-35697606) 
 wherein the entity of a tumor is identified.   
     
     
         8 . The method of  claim 7 , wherein the set of methylation markers comprises at least 240 regions, wherein the group consists of the regions listed in Table 1b. 
     
     
         9 . (canceled) 
     
     
         10 . A method comprising determining the methylation of a set of methylation markers in a sample from a patient, wherein the set of methylation markers comprises the following 10 positions: 596 (chr11, 57006229), 1717 (chr15, 28262724), 2636 (chr18, 61144199), 2805 (chr19, 46823441), 4674 (chr2, 176964685), 4999 (chr2, 225642035), 5071 (chr3, 14960020), 5576 (chr4, 13525705), 6105 (chr5, 140475760), and 6434 (chr6, 46386723). 
     
     
         11 . A method determining the methylation of a set of methylation markers in a sample from a patient, wherein the set of methylation markers comprises the following 10 positions: 650 (chr11, 64993331), 2995 (chr1, 17568007), 4233 (chr2, 50574690), 4241 (chr2, 50574708), 4428 (chr2, 111874494), 4447 (chr2, 121276804), 5537 (chr4, 1666074), 5538 (chr4, 1666075), 6524 (chr6, 83604790), and 7164 (chr7, 69971740). 
     
     
         12 . The method of  claim 1 , wherein the set of methylation markers comprises all positions listed in Table 4. 
     
     
         13 . The method of  claim 1 , wherein the lung cancer is an NSCLC selected from the group comprising adenocarcinoma and squamous cell carcinoma, or an SCLC. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the sample, is a liquid biopsy sample, or a solid tissue sample collected during surgery. 
     
     
         16 . A method comprising obtaining a liquid biopsy sample from a subject, and determining the methylation of a set of methylation markers to obtain a cell-free DNA (cfDNA) methylation signature, wherein the set of methylation markers is selected from the group consisting of the regions listed in Table 1a, 1b and 1c and comprises at least 60 regions. 
     
     
         17 . The method of  claim 15 , wherein the liquid biopsy sample is selected from the group comprising blood, plasma, serum, sputum, bronchial fluid and pleural effusion. 
     
     
         18 . The method of  claim 1 , wherein the sample is a lung biopsy sample. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A means suitable for diagnosing lung cancer, wherein the means comprises oligonucleotides which can hybridize to DNA comprising methylation markers, wherein the set of methylation markers is selected from the group consisting of the regions listed in Table 1a, 1b and 1c and comprises at least 60 regions. 
     
     
         22 . A method comprising:
 a. extracting cfDNA from the liquid biopsy sample or genomic DNA from the   solid tissue sample,   b. carrying out a bisulfite conversion,   c. producing a whole-genome bisulfite sequencing library,   d. enriching DNA regions comprising the defined methylation markers, preferably comprising contacting them with the means of  claim 21 ,   e. sequencing the enriched DNA regions,   f. aligning the sequencing data against a reference genome using the Segemehl algorithm, and   g. calculating the methylation rates.   
     
     
         23 . (canceled) 
     
     
         24 . A method for treating a lung tumor in a subject, comprising determining the methylation of a set of methylation markers in a sample from a patient according to the method of  claim 1 , detecting a pattern of methylation that identifies the presence, entity, stage, and/or prognosis of a lung tumor, and treating the subject with a suitable medicament, irradiation, or combination thereof. 
     
     
         25 . The method of  claim 24 , comprising determining the entity of a lung tumor, selecting a therapy suitable for treatment of said entity, and treating the subject with the suitable medicament, irradiation, or combination thereof. 
     
     
         26 . A method for treating a lung tumor in a subject, comprising determining the methylation of a set of methylation markers in a sample from a patient according to the method of  claim 10 , detecting a pattern of methylation that identifies the presence, entity, stage, and/or prognosis of a lung tumor, and treating the subject with a suitable medicament, irradiation, or combination thereof. 
     
     
         27 . A method for treating a lung tumor in a subject, comprising determining the methylation of a set of methylation markers in a sample from a patient according to the method of  claim 11 , detecting a pattern of methylation that identifies the presence, entity, stage, and/or prognosis of a lung tumor, and treating the subject with a suitable medicament, irradiation, or combination thereof.